Process of preparing immunoglobulin for intravenous injection by viruses double-sterilized without adding any protectant

ABSTRACT

A process of preparing a double-sterilized immunoglobulin product for intravenous injection includes dissolving Cohn&#39;s component II in distilled ice water, and adjusting to the desired pH using acetic acid. A filter is used to remove alcohol and salt and establish a desired sodium ion concentration, and the immunoglobulin concentration is adjusted to form interim product, which is bottled. The bottle is filled with gaseous carbon dioxide to achieve a pressure of 0.7 to 200 kPa, sealed in the absence of any protectant, and sterilized. The interim product is filtered, concentrated to achieve an immunoglobulin concentration of 5% to 10%, adjusted to a desired pH, filtered to remove bacteria, and stored at room temperature for 21 days. Glucose is added in order to change the osmotic pressure equilibrium of said interim product to form final product.

This application is a 371 of PCT/CN99/00081.

FIELD OF THE INVENTION

The present invention relates to a process for preparing a biologicalproduct, particularly for preparing immunoglobulin for intravenousinjection (IVIG) by using a double-sterilized method without includingany protectant.

BACKGROUND OF THE INVENTION

The production of immunoglobulin started in 1949. Since the invention ofIVIG in the 1960s, more than thirty (30) kinds of IVIG products,produced by 29 manufacturers in 12 countries, have appeared in theinternational marketplace.

Human placental blood IVIG first appeared in China in 1985, and theregulations of its manufacture and assay are recorded in “The ChinaNational Regulations of Biological Products”. In 1992, pilot-plantproduction of lyophilized low pH IVIG was approved by the Ministry ofHealth of the Peoples Republic of China.

There have been many instances of epidemic type-C hepatitis caused bythe repeated injection of large doses of IVIG Evidence has shown that itis difficult to ensure the safety of such injections without proceduresfor virus sterilization in the course of IVIG production. Since the1990s, virus sterilization, as an essential procedure in the productionof IVIG, has been regulated by many countries throughout the world.

Pasteurization was the earliest method used for virus sterilization. Forexample, virus sterilization may be accomplished in IVIG products byheating for 10 hours at 60° C. Methods such as low pH incubation, dryheat, irradiation, and filtration have also been developed to accomplishvirus sterilization. In addition, as a result of the rapid spread ofAIDS, a method using detergent to destroy the lipid-containing envelopeof a virus was developed for HIV. Presently, most IVIG products areprepared using lyophilization, in which saccharose serves as aprotectant and excipient.

Although the above-mentioned virus sterilization techniques are usefulagainst the HIV, HBV, HCV, and other viruses with common pathogenicfactors, differences in effectiveness among the various known methods ofvirus sterilization remain. Furthermore, it has been noted that a virusexisting in the presence of a protectant may be better able to survivethe sterilization process. According to recent literature (Ref. MurphyP. et al., J-Med-Virol, 41(1):61-64, 1993), although the presence of aprotectant has some effect for keeping the natural activity of aprotein, it can yield a certain protection for a virus. As a result,trace amounts of viruses may remain in the product.

In addition, organic solvents used in detergent sterilization are notcompletely recovered, which affects not only the purity of the product,but also can produce undesirable effects when the product isadministered intravenously. For example, it has been reported in severalcases that the use of saccharose as a protectant can lead to acutefailure of kidney function. (Ref. Winward D B et al., Pharmacotherapy,15(6):765-722, 1995; Hifenhaus L.& Nowak T., Vox Sang 67 (Suppl.)1:62-66, 1994; Horowitz B.et al., Blood, 86(11): 4331-4336, 1995).

Also, the clinical side effects of lyophilized IVIG outweigh thebenefits of the liquid products because the lyophilization processcauses polymerization of the IVIG molecule. Thus, the preparation ofIVIG using a lyophilization process is not favorable for large-scaleproduction.

SUMMARY OF THE INVENTION

The object of this invention is to overcome the deficiencies in thetechniques for producing IVIG that use existing methods for virussterilization. The present invention is a process for preparing abiological product, particularly for preparing immunoglobulin forintravenous injection (IVIG), by using a double-sterilized methodwithout including any protectant. Specifically, the process incorporatesa virus-sterilizing method with two different mechanisms, which serve toapproach complete virus-sterilization. As a result, IVIG may be usedmore effectively and safely in the clinical environment.

DETAILED DESCRIPTION OF THE INVENTION

The first step for preparing immunoglobulin for intravenous injection(IVIG) according to the present invention is Pasteurization. As a rawmaterial, Cohn's component II (F II) is dissolved in between andincluding 2-fold and 10-fold distilled ice water. The pH is adjusted tobetween and including 3.5 and 5.0 with an acetic acid solution having aconcentration between and including 0.2 and 2.0 mmol/L. The solution ispassed through an ultra-filter membrane having a molecular weightcut-off between and including 10 and 100 kDa, in order to remove alcoholand salt and achieve a sodium ion concentration of between and including1 and 10 mmol/L. The IVIG concentration is adjusted to between andincluding 0.5% and 2% and then bottled. The bottles are filled withgaseous CO₂ until the internal pressure is between and including 0.7 and200 kPa. Next, the bottles are sealed in the absence of any protectantand sterilized at 60° C.±1° C. for 10 hours.

The second step for preparing immunoglobulin for intravenous injection(IVIG) according to the present invention is incubation treatment at lowpH. After Pasteurization is complete, the F II solution is cleaved andpurified with an ultra-filter membrane having a molecular weight cut-offbetween and including 10 and 100 kDa. In the event that the IVIGsolution has a purity less than 97%, the purity of the solution isincreased to 97% or above using gel adsorption techniques. The IVIGsolution is then concentrated to between and including 5% and 10%. ThepH is adjusted to 4.1±0.3. The solution is then filtered to remove andstored at room temperature for 21 days. The osmotic is adjusted byadding between and including 5% and 10% Once quality is assured, theproduct may be bottled using 25 or 50 ml bottles.

In order to demonstrate the effectiveness of the present invention,virus sterilization using Pasteurization, incubation at low pH, and acombination of Pasteurization and incubation at low pH (doublesterilization method) as described in the present invention is shownusing the indicator viruses VSV, Polio-I, and Sindbis.

TABLE 1 Pasteurization - sterilizing effect on three kinds of viruses at60° C. VSV Polio-I Sinbis Log TCID₅₀/0.1 ml Log TCID₅₀/0.1 ml Log PFU/mlSterilizing control Treated control treated control treated time (h)sample sample sample sample sample sample 0 6 6.5 4 × 10⁶ 1 6 4 6.5 2 4× 10⁶   10⁴ 4 6 3 6.3 — 4 × 10⁶ <10⁴ 5 5.8 2 6.3 — 4 × 10⁶ <10³ 7 5.8 16.3 — 4 × 10⁶ <10² 10  5.8 — 6 — 4 × 10⁶ —

As shown in Table 1, the sterilizing effect of Pasteurization on thePolio-I virus is remarkable, but less so for the VSV and Sindbisviruses.

TABLE 2 Low pH incubation - sterilizing effect on two kinds of virusesat pH 4.0, 23° C. Sindbis (Log PFU/ml) Time VSV (Log TCID₅₀/0.1 ml)control treated (day) control sample treated sample sample sample 0 6.5  4 × 10⁶ 3 6.5 1   4 × 10⁵ <10⁴ 7 8 — 3.5 × 10⁶ <10³ 14  6 — 3.5 × 10⁶<10⁰ 21  5.5 — 3.5 × 10⁴ —

As shown in Table 2, the sterilizing effect of low pH incubationdemonstrates that VSV is sensitive to an acidic environment of pH 4.0because it was rendered inactive on the seventh day after treatment. Thesterilizing effect of low pH incubation demonstrates that Sindbis isless sensitive to the same acidic environment because it was renderedinactive on the twenty-first day after treatment.

TABLE 3 Double sterilization - sterilizing effect on three kinds ofviruses VSV Polio-I Sindbis Log TCID₅₀/0.1 ml Log TCID₅₀/ml Log PFU/mlSterilizing control treated control treated control treated time (h)sample sample sample sample sample sample 60° C. 0 6 6.5   4*10⁶ heating5 6 2 6.5   4*10⁶ 10³ Stored at 10  6 — 6.3 —   4*10⁶ — pH 4.0  7 days5.5 — 6 — 3.5*10⁶ — 23° C. 14 days 5.5 — 6 — 3.5*10⁶ — 21 days 5 — 5.5 —3.5*10⁶ —

As shown in Table 3, the effect of double sterilization demonstratesthat all three indicator viruses have been inactivated after 10 hours ofPasteurization.

In order to further demonstrate the effectiveness of the presentinvention, the National Institute for Control of Pharmaceutical andBiological Products and AIDS Detection and Confirmation Laboratory ofthe People's Liberation Army (PLA) obtained the following results afterconducting a separate series of tests. These are shown in Table 4 andTable 5.

TABLE 4 Pasteurization - sterilizing effect on four kinds of viruses at60° C. VSV Polio-I HIV Sindbis Log TCID₅₀/0.1 ml Log TCID₅₀/0.1 ml LogTCID₅₀/0.1 ml Log PFU/ml Time control treated control treated controltreated control treated (h) sample sample sample sample sample samplesample sample 0 7.00 5.00 6.00 3.13   6.00   5.77 — 6.63 0.5 ≦−0.50≦0.50 ≦−0.50 ≦0.50 — — — ND 1 ≦−0.50 ≦0.50 ≦−0.50 ≦0.50 — — — ND 4≦−0.50 ≦0.50 ≦−0.50 ≦0.50 — — — ND 6 ≦−0.50 ≦0.50 ≦−0.50 ≦0.50 — — — —10 ≦−0.50 ≦0.50 ≦−0.50 ≦0.50 — — — ND ≦5.80 <2.00 — —

In this experiment, samples were treated by Pasteurization, which wasaccomplished by heating the samples at 60° C. for 10 hours. As shown,there was substantial sterilization for the indicator viruses VSV, HIV,Sindbis, and Polio-I. After heating for 30 minutes, the virus titerswere decreased below the detection limit. The viruses that experienceddecreases were VSV: ≧4.50-4.63 log TCID₅₀/0.1 ml, Polio-I: ≧2.63-3.63log TCID₅₀/0.1 ml, HIV: ≧3.77-4.17 log TCID₅₀/0.1 ml, and Sindbis:≧6.32-6.41 log PFU/ml, respectively. For the samples in which heatinglasted 6 hours and 10 hours, three generations of blind passage werenegative. The virus minimal detection limits were: ≦0.50 Log TCID₅₀/0.1ml, titer of Sindbis basal virus: 7.70 log PFU/ml. An “ND” indicatesthat no virus was detected.

TABLE 5 Low pH incubation - sterilizing effect on three kinds of virusesat room temperature VSV HIV* Log TCID₅₀/ Log TCID₅₀/ Sindbis Time 0.1 ml0.1 ml Log PFU/ml (days) Acid Neutral Acid Neutral Acid Neutral 0 3.886.38   5.77 6.00 3.75 7.34 3 3.13 6.13 — — ND 7.09 7 1.75 5.88 <2.005.80 ND 7.09 14  ≦0.50 5.18 — — ND 6.60 21  ≦0.50 4.88 — — ND 6.23

In this experiment, samples were incubated at a pH of 4.0±0.2 and atemperature of between 22 and 24C. for 21 days. The viruses thatexperienced decreases were VSV: ≧5.88-6.63 log TCD₅₀/0.1 ml, HIV:≧3.77-4.17 log TCD₅₀/0.1 ml, and Sindbis: ≧7.33-7.74 log PFU/ml. Threegenerations of blind passage were negative. The virus minimal detectionlimits were: ≦0.50 Log TCID₅₀/0.1 ml, titer of Sindbis basal virus: 8.35log PFU/ml. An “ND” indicates that no virus was detected. An “*”indicates that a sample after Pasteurization was used.

In summary, the following conclusions may be drawn from the experimentsdescribed above:

1. Pasteurization at 60° C. for 10 hours is capable of sterilizing fourkinds of indicator virus. This is particularly effective for VSV andPolio-I viruses.

2. Low pH incubation provides a strong sterilizing effect on VSV, HIV,and Sindbis viruses, which were completely inactive in 7 days.

3. Double-sterilization, which includes Pasteurization for 10 hoursfollowed by room temperature incubation at pH 4.0 for 21 days,demonstrated that all indicator viruses are inactivated more thoroughly.

As compared to existing techniques, the advantages, as described herein,for preparing immunoglobulin for intravenous injection (IVIG) accordingto the present invention by using a double-sterilized method withoutincluding any protectant are as follows:

1. The process described by the present invention does not incorporateany protectant constituent. As a result, the process effectivelyinactivates viruses while maintaining the integrity of the IVIG moleculeand ensuring the natural biological activity, purity, and safety of theproduct.

2. The process described by the present invention allows for theliquidus preparation of IVIG, rather than a lyophilized powder process.As a result, the process shortens production time, reduces energyconsumption, and favors large-scale production while maintaining astable and reliable product.

3. The process of the present invention incorporates glucose in theproduct, which adjusts the osmotic pressure equilibrium of the solution.As a result, this offers certain patients relief from undesirableeffects after IVIG is administered.

EXAMPLES Example 1

The first step for preparing human blood IVIG using thedouble-sterilization without including any protectant method of thepresent invention is Pasteurization. A 16.5 kg F II precipitate isdissolved in 165 liters of distilled water at 0° C. The pH is adjustedto 3.5 using a 1 mmol/liter acetic acid solution. The solution is passedthrough an ultra-filter membrane having a molecular weight cut-off of100 kDa, in order to remove alcohol and salt and achieve a sodium ionconcentration of 1.2 mmol/liter. The immunoglobulin concentration isadjusted to 1.5% and the solution is bottled and sealed. The internalpressure is adjusted to 100 kPa by charging with carbon dioxide. Thesolution is then sterilized at 60° C.±1° C. for 10 hours.

The second step is an incubation treatment at low pH. AfterPasteurization, the F II solution is cleaved and purified with anultra-filter membrane having a molecular weight cut-off of 100 kDa. Inthe event that the immunoglobulin solution has a purity of less than97%, the purity of the solution may be increased to greater than orequal to 98% using DEAE-Sephadex A-50 gel adsorption. The immunoglobulinis concentrated to 5% and the pH is adjusted to 4.1. The solution isthen filtered in order to remove bacteria. The solution is then storedfor 21 days after which 5% glucose is added. The product is then testedfor its physical and chemical properties including pyrogen and sterilitytests according to the regulations governing human blood immunoglobulin.If the product passes the tests, it is filtered to remove bacteria andbottled in 25 or 50 ml bottles.

Example 2

The first step for preparing human blood IVIG using thedouble-sterilization without including any protectant method of thepresent invention is Pasteurization. A 15 kg F II precipitate isdissolved in 150 liters of distilled water at 0° C. The pH is adjustedto 5.0 using a 0.2 mmol/liter acetic acid solution. The solution ispassed through an ultra-filter membrane having a molecular weightcut-off of 10 kDa, in order to remove alcohol and salt and achieve asodium ion concentration of 10 mmol/liter. The immunoglobulinconcentration is adjusted to 2% and the solution is bottled and sealed.The internal pressure is adjusted to 0.7 kPa by charging with carbondioxide. The solution is then sterilized at 60° C.±1° C. for 10 hours.

The second step is an incubation treatment at low pH. AfterPasteurization, the F II solution is cleaved and purified with anultra-filter membrane having a molecular weight cut-off of 10 kDa. Inthe event that the immunoglobulin solution has a purity less than 97%,the purity of the solution may be increased to greater than or equal to98% using DEAE-Sephadex A-50 gel adsorption. The immunoglobulin isconcentrated to 8% and the pH is adjusted to 4.1. The solution is thenfiltered in order to remove bacteria. The solution is then stored for 21days after which 10% glucose is added. The product is then tested forits physical and chemical properties including pyrogen and sterilitytests according to the regulations governing human blood immunoglobulin.If the product passes the tests, it is filtered to remove bacteria andbottled in 25 or 50 ml bottles.

Example 3

The first step for preparing human blood IVIG using thedouble-sterilization without including any protectant method of thepresent invention is Pasteurization. A 16.5 kg F II precipitate isdissolved in 165 liters of distilled water at 0° C. The pH is adjustedto 4.2 using a 2.0 mmol/liter acetic acid solution. The solution ispassed through an ultra-filter membrane having a molecular weightcut-off of 50 kDa, in order to remove alcohol and salt and achieve asodium ion concentration of 5 mmol/liter. The immunoglobulinconcentration is adjusted to 0.5% and the solution is bottled andsealed. The internal pressure is adjusted to 200 kPa by charging withcarbon dioxide. The solution is then sterilized at 60° C.±1° C. for 10hours.

The second step is an incubation treatment at low pH. AfterPasteurization, the F II solution is cleaved and purified with anultra-filter membrane having a molecular weight cut-off of 50 kDa. Inthe event that the immunoglobulin solution has a purity less than 97%,the purity of the solution may be increased to greater than or equal to98% using DEAE-Sephadex A-50 gel adsorption. The immunoglobulin isconcentrated to 10% and the pH is adjusted to 4.1. The solution is thenfiltered in order to remove bacteria. The solution is then stored for 21days after which 8% glucose is added. The product is then tested for itsphysical and chemical properties including pyrogen and sterility testsaccording to the regulations governing human blood immunoglobulin. Ifthe product passes the tests, it is filtered to remove bacteria andbottled in 25 or 50 ml bottles.

What is claimed is:
 1. A process of preparing a double-sterilizedimmunoglobulin without protectant product for intravenous injectioncomprising: dissolving Cohn's component II in between and including2-fold and 10-fold distilled ice water; adjusting to a pH of between andincluding 3.5 and 5.0 with between and including 0.2 and 2.0 mmol/literacetic acid; filtering with an ultra-filter membrane having a molecularweight cut-off of between and including 10 kDa and 100 kDa to removealcohol and salt and establish a sodium ion concentration of between andincluding 1 and 10 mmol/liter; adjusting the immunoglobulinconcentration to between and including 0.5% and 2% to form interimproduct; adding said interim product to a bottle; filling said bottlewith gaseous carbon dioxide to achieve a pressure of between andincluding 0.7 and 200 kPa; sealing said bottle in the absence of anyprotectant; sterilizing said bottle containing said interim product at atemperature between and including 59° C. and 61° C. for 10 hours;filtering said interim product with an ultra-filter membrane having amolecular weight cut-off of between and including 10 kDa and 100 kDa;concentrating said interim product to achieve an immunoglobulinconcentration of between and including 5% and 10%; adjusting to a pH ofbetween and including 3.8 and 4.4; filtering said interim product toremove bacteria; storing said interim product at room temperature for 21days; and adding between and including 5% and 10% glucose in order tochange the osmotic pressure equilibrium of said interim product to formfinal product.
 2. The process of claim 1, comprising an additional stepafter filtering said interim product with an ultra-filter membranehaving a molecular weight cut-off of between and including 10 kDa and100 kDa of applying said interim product to a gel adsorption medium inorder to improve purity.